1. Field of the Invention
The invention is directed to a laser scanning microscope (LSM) with a laser diode.
2. Description of Related Art
Laser scanning microscopes (LSM) are used for confocal recording of images of a sample by a laser beam which usually scans in a zigzag shape. Prior to a scanning process, individual regions to be recorded (regions of interest, or ROI) can be determined. To avoid unnecessary stress on the sample, the laser light should be switched on as accurately as possible upon entering the region to the scanned. This is also true for the peripheral regions of the sample, which are generally not to be imaged, in the area where the laser beam changes direction because the speed at which the laser beam moves over the sample is slowest in this area and the radiation loading is therefore at its highest. In addition to processes for switching on and switching off from and to the zero level, changes in the beam intensity between two intensity levels other than zero are also required depending on the application.
In the prior art, the light intensity of a laser beam of a diode laser in an LSM can be controlled with high accuracy by means of an acousto-optic component, for example, an AOM (acousto-optic modulator) or an AOTF (acousto-optic tunable filter). This is disclosed, for example, in DE 197 02 753 A1. However, a component of this kind requires a relatively large installation space and is costly.
In order to dispense with an acousto-optic component while nevertheless enabling adjustment of the light intensity on the sample, the optical output power of the laser diode can be controlled directly by changing the electric current. In this way, both the attenuation function and the modulation function can be achieved in a manner analogous to the use of an acousto-optic component.
However, laser diodes which are modulated directly by diode current have drawbacks with respect to imaging. The most severe drawback is that important laser parameters become unstable when it becomes necessary in a certain application to reduce the diode current in the range of the laser threshold. Particularly noteworthy parameters are the polarization, which can fall from values greater than 100:1 to well below 10:1, and the spectral width of the laser beam—the diode transitions seamlessly from laser mode to LED mode so that the spectral width can increase to several nanometers. Stability and power calibration are also critical because the diode becomes unstable in the current intensity range around the laser threshold, which leads to excessive noise. Further, the threshold current exhibits a variation over the lifetime of the laser diode so that it is necessary to calibrate the power in the threshold range at regular intervals. Moreover, a change in current through the laser diode within the framework of direct modulation, also above the laser threshold, results in a slight shift of the center wavelength of the emitted spectral line. This can lead to a change in the beam direction due to the dispersion of the prisms used for beam shaping or in the microscope M or scanning unit S, which impairs the accuracy of exposure.
Another application of a LSM is fluorescence lifetime imaging (FLIM). In this case, short laser pulses of durations typically from 20 ps to 100 ps are used as illumination. In the prior art, these short laser pulses are generated by means of a laser diode controlled by electric pulses, so-called gain switching. In so doing, the electric pulses are adapted to the respective individual laser diode so as to generate optimal optical pulses (i.e., with a full width (FWHM) from 20 ps to 100 ps, steep edges, insignificant or nonexistent shoulders, and no long afterglows). Changes in intensity are caused by neutral density glass because changes in intensity due to a change in the electric control parameters (e.g., the electric pulse height and pulse width or CW bias current) necessarily entail changes in the optical pulse shape and can therefore be carried out only within a narrow, carefully selected range.